Pseudomonas putida infection induces immune-antioxidant, hepato-renal, ethological, and histopathological/immunohistochemical disruptions in Oreochromis niloticus: the palliative role of titanium dioxide nanogel

Background Pseudomonas putida is a pathogenic bacterium that induces great losses in fishes, including Nile tilapia (Oreochromis niloticus). Currently, the application of nanomaterials in aquaculture practices has gained more success as it endows promising results in therapies compared to traditional protocols. Objective Therefore, the current perspective is considered the first report to assess the anti-bacterial efficacy of titanium dioxide nanogel (TDNG) against Pseudomonas putida (P. putida) in Nile tilapia. Methods The fish (n = 200; average body weight: 47.50±1.32 g) were allocated into four random groups (control, TDNG, P. putida, and TDNG + P. putida), where 0.9 mg/L of TDNG was applied as bath treatment for ten days. Results Outcomes revealed that P. putida infection caused ethological alterations (surfacing, abnormal movement, and aggression) and depression of immune-antioxidant variables (complement 3, lysozyme activity, total antioxidant capacity, superoxide dismutase, and reduced glutathione content). Additionally, a substantial elevation in hepatorenal biomarkers (aspartate and alanine aminotransferases and creatinine) with clear histopathological changes and immuno-histochemical alterations (very weak BCL-2 and potent caspase-3 immuno-expressions) were seen. Surprisingly, treating P. putida-infected fish with TDNG improved these variables and obvious restoration of the tissue architectures. Conclusion Overall, this report encompasses the key role of TDNG as an anti-bacterial agent for controlling P. putida infection and improving the health status of Nile tilapia. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-024-03972-6.


Introduction
In aquaculture, the tilapia industry is one of the most stable and steady-growing.Nile tilapia (Oreochromis niloticus) is the best-ranked and most substantial tilapia species being cultivated and traded [1].With rapid progress and intensification in the aquaculture industry, various emerging diseases have arisen [2].In particular, bacterial infections induce hazardous effects on fish, including septicemia, hemorrhage, and mortalities [3,4].
Among bacterial diseases, the Pseudomonas species is one of the most virulent pathogens that invades fish and results in ulcerative syndrome [5].Pseudomonas putida is a highly pathogenic bacterium that infects Nile tilapia and induces ascites, exophthalmia, and ulcers in the body [6].P. putida is an opportunistic Gram-negative pathogen related to the Pseudomonaceae family that normally exists in the aquatic ecosystem.It is found in healthy fish as a part of the normal gut microflora [7,8].A recent study reports that P. putida causes higher mortalities in Nile tilapia as it carries virulence-linked genes (ToxA, Nan1, and ExoS) [9].
The emergence of drug-resistant bacteria increases the demand for alternative strategies to treat bacterial infections.One of the successful strategies is utilizing nano-based materials as anti-bacterial agents [10,11].The nanomaterials are utilized as antimicrobial agents because of their power to penetrate the membranes of the bacteria and disrupt the formation of biofilm [12].In particular, metallic nanoparticles (NPs) are attracting more attention owing to their great success in pharmaceutical and biological applications [13,14].Various metal oxide-NPs exhibited potent antimicrobial activity, like titanium, magnesium, zinc oxide, copper, and silicon oxides.They are characterized by many features, including heat resistance and less toxicity, and reveal strong activity against resistant strains of many microorganisms [15,16].Also, they can be utilized as mineral element supplements which are essential for nourishing cells [17].Nano-based titanium oxide elicited potent inhibitory activity against the growth of bacteria because of its little nano-sized and strong oxidizing activity [18].Nanogels (NGs) are a recent and superior scheme for diagnosing and treating a broad spectrum of diseases [19].Due to their small size, NGs assist as significant drug nano-carriers, which can penetrate tissues in a transcellular way [20].The gel-derived titanium dioxide (TiO 2 ) has a tested anti-bacterial activity [21].Recently, it has proved the effective inhibitory activity of TiO 2 nano-form against 15 species of bacteria [22].Current studies apply other types of NGs in aquaculture practice and prove great success in alleviating toxicity and promoting immune-antioxidant function [23,24].
Hence, the present report is a pioneering trial to investigate the anti-bacterial impact of TiO 2 nanogel (TDNG) as a watery addition on the health status of Nile tilapia challenged with P. putida.The investigation included assaying the fish behavior, immunity, antioxidant activity, and histological architectures.This gives a theoretical basis for the logical application of TDNG.

TDNG synthesis and characterization
Firstly, the synthesis of titanium dioxide nanoparticles (TiO 2 NPs) was achieved through a simple sono-chemical method [25].0.25 g of TiO 2 (Sigma-Aldrich Co., MO, USA) was added to 100 mL of 10 M NaOH (El Naser Chemical Co., Egypt) in a 250 mL flask.The solution was then subjected to ultrasonic waves (Sonica 4200 EPS3, Milano, Italy) under the condition of 88% amplitude and 0.82 cycles for 1.5 h at room temperature with adjusting pH to 7.0 using 0.1 M HCL (El Naser Chemical Co., Egypt).The resulting TiO 2 NPs solution was then centrifuged three times using double distilled deionized water for washing.
To synthesize TDNG as TiO 2 NPs/carbopol hybrid nanogel, 0.2 g of TiO 2 NPs was dispersed in 40 mL of ethanol (95%) and added to 0.4 g of carbopol dissolved in 40 mL of ethanol (95%).The resulting mixture was stirred using a mechanical stirrer for 65 min.Then, 1.2 mL of trimethylamine was added drop by drop, and the stirring continued for another 65 min.until a white gel was obtained.TDNG was prepared in low and high-viscosity forms.Characterization procedures were divided into three categories: morphology, index, and identification [26].

Acclimation of fish and ethical approval
The experimental design of the current study was approved by the Institutional Animal Care and Use Committee at Zagazig University in Egypt (ZU-IACUC/2/F/333/2022). Nile tilapia (47.50 ±1.32 g) were sorted from a private fish farm in Al-Abbassa, Sharkia Governorate, Egypt with prior informed consent from the owners.For acclimation, fish were maintained for 2 weeks in 100 L of well-aerated aquaria (ten fish/ aquarium).The excretory wastes were disposed of daily through siphoning.The fish received a commercial diet at 3% of their body mass twice daily throughout the acclimation period.The physio-chemical biomarkers (temperature, dissolved oxygen, ammonia, and pH) of the rearing water were monitored daily according to APHA [27] guidelines, and they were 23.00±2 °C, 6.50 ± 0.11 mg/L, 0.01 ± 0.03 mg/L, and 7.30 ± 0.12, respectively.

Assessing the initial concentration of TDNG
Fish (n = 70) were exposed to 7 different concentrations (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mg/L) of TDNG for ten days to establish the initial concentration for the treatment trial.Each group was kept in 100 L well-aereated aquarium (10 fish/aquarium).Weighed amounts of TDNG was dissolved in initial amount of distilled water (5 mL) before adding to the aquarium water to obtain the final concentrations.Daily records of the clinical observations were kept throughout the initial study.TDNG concentrations were deemed safe between 0.3 and 1.2 mg/L, with 0.9 mg/L being the concentration employed for treatment (Supplementary Table 1).

Bacterial strain (P. putida)
The current perspective was carried out on P. putida, formerly isolated from diseased Nile tilapia (Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Zagazig University).It was recognized by the VITEK 2-C15 automated system for bacterial identification (BioMérieux, Craponne France) and conventional biochemical assays following the manufacturer's instructions as documented by Scheidegger et al. [28] and Zhou et al. [29] at the Department of Microbiology and Immunology, National Research Centre (NRC), Dokki, Giza, Egypt.P. putida was streaked onto pseudomonas agar base (Oxoid, England) and incubated for 24 h at 37 °C.One colony was taken to incubate in brain heart infusion broth (Sigma-Aldrich) for 24 h at 37 °C.After centrifuging the cultured broth at 4 °C for 10 min at 3000 ×g, the pellet was retrieved and suspended in sterile phosphatebuffered saline (PBS).
The lethal dose (LD 50 ) of P. putida was established.Fish (n=80) were distributed into four groups in duplicates (10 fish/ replicate; 20 fish/group).Fish were given intraperitoneally (IP) different doses of a live, 24 h-old P. putida culture suspension (10 6 -10 9 CFU/fish).Another ten fish (control group) were IP given with 0.1 mL sterile saline.The mortality of fish was then noted four days following the inoculation.According to the Probit Analysis Program, version 1.5 (US Environmental Protection Agency), the LD 50 was 3.9 × 10 8 CFU/mL.A sub-lethal dose of 1.5 × 10 8 CFU/mL was used in the trial.

Experimental protocol
For ten days, fish (n = 200) were randomized into four groups in five replicates (10 fish/replicate).The groups were the control (no TDNG addition or P. putida challenge), TDNG, P. putida, and TDNG + P. putida groups.The fish of P. putida and TDNG + P. putida groups were IP injected with 0.1 mL of P. putida (1.5 ×10 8 CFU/mL).After the onset of clinical signs, 0.9 mg/L of TDNG was added to the aquarium water, and this procedure was continued for ten days.The excretory wastes were disposed daily through siphoning and complete water exchange was performed three times weekly.To maintain the applied TDNG concentration after water exchange, the freshly made TDNG solution was added.During the trial's ten-day run, daily records of clinical signs and mortality were kept.

Behavioural investigations
The behavior patterns were recorded in all experimental groups at a fixed time once daily (8-9 a.m.) throughout the experimental period by direct observation technique using a video camera and stopwatch [30].The behavior frequencies were observed for 15 min.intervals (5 min/aquarium).The recorded behavior categories were foraging, swimming [31], surfacing [32], resting [33], and abnormal movement [34].Moreover, aggressive behaviour was recorded, including approach, chasing, fleeing, fin tugging, butting, and mouth pushing [35].

Sampling
On the last day of the ten-day experiment, fish (15 fish per group) were randomly picked to drain samples.Based on Neiffer and Stamper [36] method, fish were anesthetized using a benzocaine solution (100 mg/L), and then blood was collected from the caudal blood vessels using tubes without anticoagulant.Centrifugation was carried out for the samples at 1750 × g for 10 min after incubation at 22±2 °C for 5 h.Pure serum was then maintained at 20 °C to assess biochemical and immunological parameters.Hepatic and renal samples were collected for antioxidant, histopathological, and immunohistochemical assessments.
The immune biomarkers in serum, including complement 3 (C3) and lysozyme (LYZ) activities were assessed.Both of them play a substantial role in the innate immune function of fish against infection.The C3 was determined by immuno-turbidimetry using Cusabio kits (CUBIO Innovation Center, Houston, USA) with a Catalog No (CSB-E09727s) following the manufacturer protocol.Meanwhile, the level of LYZ was measured using the inhibition zone method in agarose gel plates [37].

Histopathological and immunohistochemical investigation
Hepatopancreas and kidney samples were collected from all experimental groups, fixed using 10% buffered neutral formalin, then dehydrated using ascending degrees of alcohol, cleared in xylene, and finally soaked in paraffin.Paraffin sections (5 μm in thickness) were cut and stained using hematoxylin and eosin (H&E) for general histology and then inspected by an optical microscope fitted with camera system (Olympus BX53), following a prior method [41].
The immunohistochemical (IHC) assay was conducted in the de-paraffinized section (5 μm thick) with an in situ cell apoptosis identification kit (MK1020, Boster, China) according to the manufacturer's guidelines, as reported [42].B-cell lymphoma 2 (BCL-2) and caspase-3 were identified by IHC.For this reason, concisely, an HRP/ DAB detection IHC kit (ab80436 Abcam, China) was utilized based on the manufacturer's method.The sections were exposed to de-paraffinization, and then, sections carrying formalin-fixed paraffin tissues were rehydrated.A block of hydrogen peroxide was included to protect the sections, then incubated for 10 min.Following antigen retrieval (100 × Citrate Buffer, ab64236 Abcam, China) for 20 min, the sections were exposed to immunoreaction over-night at 4 °C using 10 μg/mL primary antibodies (AB-20074b and AB20158b, Sangon Biotech, China) counter to BCL-2 and caspase-3 in case of negative controls, the sections were dipped in PBS as a substitute of the definite antibody.Then, mouse-specific HRP was used for conjugation and incubation for 15 min at room temperature.DAB was used for the tissue sections that were counterstained using hematoxylin.

Analysis of data
Via Shapiro-Wilk normality, all collected data were examined for norm homogeneity.Following that, the data were statistically analyzed using one-way-ANOVA (analysis of variance test) using SPSS version 22 (SPSS, Richmond, VA, USA).Tukey's range test was conducted to evaluate the differences between means at a 95% confidence level.The means ± standard error (SE) were used to present the data.To investigate the survival probability of fish in each group, the Kaplan-Meier model was applied.Moreover, the Mantel-Cox (log-rank) test was applied to see if there were any variations between the groups.

Characterization of TDNG
Figure 1 : displays different classes of TDNG characterization.The morphology class included surface morphology and particle shape that was retrieved in atomic force microscopy (AFM; Fig. 1A), scanning electron microscopy (SEM; Fig. 1B), and transmission electron microscopy (TEM; Fig. 1C) images.They exhibited that the TDNG had a spherical shape.The index class (size and surface charge) presented in zeta potential (Fig. 1D) and dynamic light scattering images (DLS; Fig. 1E).The data revealed that the particles had a very good colloidal nature in aqueous solution, as indicated by the high zeta potential value of -41mV.Also, the particles were homogeneous in size (with only one peak in the DLS chart), with a size of approximately 22 nm.
The identification class appeared in the X-ray diffraction image (XRD; Fig. 1F).It verified that no secondary phases were necessary for the synthesis process to be valid.However, there were no distinctive peaks because the gel creation was amorphous.

Behavioral alterations
Table 1 shows the different behaviors recorded during the trial (ten days).There were no behavioral alterations recorded in the control and TDNG groups.P. putida infection caused a significant reduction in foraging (p<0.05) and an increase in the abnormal swimming, surfacing, resting, aggression traits (approach, chasing, fleeing, and mouth pushing), and abnormal movement (circular and vertical) as compared with control.On the contrary, treatment of infected with TDNG (TDNG + P. putida group) markedly enhanced (p<0.05) the foraging and declined the other behaviors compared with the infected one without treatment (P.putida group).The other aggressive traits (fin tugging and butting) did not significantly alter between groups.hemorrhages, fin rot, and severe skin ulcerations (Fig. 2B  and C).Contrarily, administration of 0.9 mg/L TDNG to infected fish (TDNG + P. putida) improved the previous clinical signs.However, some fish showed skin darkness and redness of the caudal fin with fin rot (Fig. 2D).

Biochemical variables
Between the control and TDNG groups, there are no discernible variations in the values of AST, ALT, or creatinine (p > 0.05), according to Fig. 4. The P. putida group showed a considerable increase (p<0.0001) in these variables when compared to the control.The TDNG + P. putida group showed a markedly significant drop (p<0.0001) in these variables compared to the P. putida group.

Immune-antioxidant variables
As shown in Table 2, the level of C3 and LYZ (immunological indicators) and TAC, SOD, and GSH (antioxidant markers) in the TDNG group markedly increased (p<0.0001)compared with control.These variables of the P. putida group showed a significant decrease (p<0.0001)relative to the control.In contrast to the P. putida group, there was a significant improvement (p<0.0001) in these markers in the treated group (TDNG + P. putida).

Histopathological findings
The hepato-pancreas of the control group showed normal histo-architectures of hepatic cells and pancreatic acini (Fig. 5A).As well, the hepatic parenchyma of the TDNG group were vacuolated due to fat or glycogen storage beside preserved pancreatic acini (Fig. 5B).While, focal areas of degenerated and necrotic hepatocytes, few apoptotic areas of pancreatic acini, and congested portal vein were the most encountered lesions observed in the hepato-pancreas of P. putida group (Fig. 5C).These alterations were decreased in the TDNG + P. putida group, where dilated portal vein with maintain architectures of hepatocytes and pancreatic acini were demonstrated (Fig. 5D).Moreover, the kidneys of the control and TDNG groups displayed normal cytoarchitecture of glomerular tufts and renal tubules (Fig. 6A and B).While P. putida induced (Fig. 6C) the presence of necrotic Fig. 4 The biochemical variables of Nile tilapia experimentally infected with Pseudomonas putida and exposed to titanium dioxide nanogel (TDNG) (0.9 mg/L) for ten days.A Aspartate aminotransferase (AST; U/mL, p<0.0001).B Alanine aminotransferase (ALT; U/mL, p<0.0001).C Creatinine (mg/ dL, p<0.0001)Table 2 The immune-antioxidant variables of Nile tilapia experimentally infected with Pseudomonas putida and exposed to titanium dioxide nanogel (TDNG) (0.9 mg/L) for ten days tubules and some atrophied glomeruli.In addition, the kidney of the TDNG + P. putida group exhibited hyaline globules within a few numbers of the renal epithelium; meanwhile, an apparent normal majority of renal parenchyma was seen (Fig. 6D).

Immunohistochemical findings
Immune-staining levels against BCL-2 appeared as brown granules in the hepatopancreas and kidney sections were markedly demonstrated in the control (Figs.7A and 8A) and TDNG groups (Figs.7B and 8B), respectively.P. putida infection induced an obvious reduction of BCL-2 expression in the hepatopancreas tissue (Fig. 7C) and negative expression in the kidney tissue (Fig. 8C).A mild to moderate number of immune-positive cells were obvious in the hepatopancreas (Fig. 7D) and kidney tissue (Fig. 8D) of the TDNG + P. putida group.Figures 9 and 10 exhibited stained hepatopancreas and kidney sections against caspase-3, where undetectable immunostained cells in the control (Figs.9A and 10A) and TDNG groups (Figs.9B and 10B), respectively, were seen.Diffusely cytoplasmic expressions of caspase-3 in a wide number of hepatopancreas (Fig. 9C) and renal tubule cells (Fig. 10C) were observed in the P. putida group.Contrarily, the hepatopancreas (Fig. 9D) and kidney tissues (Fig. 10D) of the TDNG + P. putida group exhibited few positive immunostained cells.

Discussion
Recent research has shown that applying nanomaterials in aquaculture fields enhances fish performance and health status [43,44].In the last decades, TiO 2 NPs have been successfully applied as they own a proven toxic mechanism against bacteria [45].Based on many studies, it is opined that metal oxides have positive charges.
Meanwhile, the microorganism carries negative charges; this induces electromagnetic attraction between the metal oxides and the microorganisms, causing oxidization and death of microorganisms [46].Therefore, the current perspective is based primarily on assessing the efficacy of aqueous exposure of TDNG on behavior, hepato-renal functions, immune-antioxidant capacity, In evaluating the ethological changes, our study clarified that P. putida challenge caused various behavioral alterations (abnormal swimming, surfacing, resting, and aggression), clinical signs (skin darkness, body hemorrhages, and severe skin ulcerations), and lower survivability (56%).This could be dominated by the existence of virulence genes [exotoxin A (tox A), nan1, and the exoenzyme S (exo S)], which hasten the infection process.Salama and Gharib [6] and Enany et al. [47] identified similar outcomes in Nile tilapia.
Exposure of the P. putida-infected fish to TDNG revealed a noticeable improvement in clinical signs and behaviors and a marked increase in fish survivability (84%).This could be dominated by the direct antibacterial action of nano-sized TDNG on the cell wall of P. putida.Earlier studies supported our findings and reported an interaction between the positive charge of TiO 2 and the negative charges of the bacterial cells.Consequently, electromagnetic power was produced between metal oxide surfaces and bacterial cells.Moreover, TiO 2 produces ions that can interact with the -SH group of proteins that limit the movement of material, lessening their permeability [45,48].
C3 and LYZ have a substantial role in the innate immune function of fish against infection [49,50].Caspase-3 means extrinsic apoptosis which is a development of cell death that plays a crucial role in the homeostasis of tissues [51].Meanwhile, intrinsic apoptosis is the response to a stimulatory reaction mediated by the interaction of the BCL-2 family and its membranes [52].Interestingly, the current study displayed the immunosuppressing activity of P. putida expressed by a marked decline in the C3 and LYZ values, plus alterations in immuno-histopathological parameters (down-regulation of BCL-2 and strong caspase-3 immune reaction).This could be dominated by the direct action of the virulence genes which leads to cytotoxicity, and accordingly immune dysfunction [53].Concurrently, Alzahrani et al. [9] revealed that P. putida inhibited the immune biomarkers.The basic attention is devoted to the immune-modulating activity of TDNG following exposure to the bacterial infection (P.putida) which is indicated by augmenting levels of C3 and LYZ plus up-regulation of the response of the BCL-2 and down-regulation of caspase-3.It is opined that TiO 2 has a strong anti-bacterial action that can directly suppress bacterial activity by its nano-size [12,54], inducing a remarkable improvement in the immune parameters and accordingly, strengthens the immune system.
The crucial antioxidant biomarkers, including TAC, SOD, and GSH, have a deep-rooted role in mitigating oxidative damage in the body via relapsing free radicals and reflecting antioxidant-defending activity.Oxidative stress is diligently associated with the incidence and progress of a disease, and when the body's antioxidant mechanism is unbalanced, it could produce oxidative damage in pathological states [55].Therefore, it is essential to assess the antioxidant capacity, hepato-renal function, and histopathological alterations in response to exposure to NGs and bacterium to reflect the antioxidant status of the fish.The present study clarified the occurrence of oxidative damage in the hepatopancreas and kidneys in the infected group in P. putida, indicated by a reduction in the hepatic antioxidant biomarkers (TAC, SOD, and GSH), elevating hepatic and kidney function biomarkers (ALT, AST, and creatinine), and remarkable histopathological changes in hepato-renal tissues.The weakened antioxidant system and oxidative damage may be caused by increased reactive oxygen species (ROS) release in the cell membrane brought on by P. putida toxins [56].Likewise, a recent study by Alzahrani et al. [9] documented that P. putida suppresses the activities of antioxidant indices (SOD and catalase).
However, the exposed group to TiO 2 pronounced antioxidant and anti-bacterial activities indicated by an increase in the antioxidant parameters, a modulation in the hepato-renal biomarkers, and regeneration in the architecture in the hepato-renal tissues.An earlier study supported our findings and explained the antioxidant-antibacterial activity of TiO 2 via inducing a sudden decrease in the integrity of the bacterial cell membrane plus ROS release where superoxide species is produced to degrade the biomolecules [57].Similarly, Abdel Rahman et al. [23] reported a clear improvement in the architecture in the hepatic and renal tissues post-exposure of African catfish (Clarias gariepinus) to magnetite nanogel.Also, Mahboub et al. [24] displayed the strong antioxidant activity of chitosan nanogel indicated by modulating values of SOD and catalase.

Conclusions
Based on the study outcomes, TDNG at a concentration of 0.9 mg/L is a versatile anti-bacterial tool against P. putida infection.It can decrease mortality, oxidative stress, and hepato-renal malfunction in P. putida-infected fish.Plus, it can be utilized as an immunomodulatory and antioxidant agent as it promotes activity on immune-antioxidant parameters and regenerates the histopathological changes induced by bacterial infection.Further studies are required to assess the dietary intervention of TDNG and test its influence on other fish species.

Figure
Figure 2A displays no obvious clinical signs in the control and TDNG groups.P. putida infection induced various clinical signs, including skin darkness, body

Fig. 2 Fig. 3
Fig.2The clinical signs of Nile tilapia experimentally infected with Pseudomonas putida and exposed to titanium dioxide nanogel (TDNG) (0.9 mg/L) for ten days.A Control and TDNG fish display a normal appearance.B & C Fish of the P. putida group display skin darkness, body hemorrhages, fin rot (black arrows), and severe skin ulcerations (yellow arrow).D Fish of the TDNG + P. putida group displaying skin darkness, redness of the caudal fin, and fin rot (black arrow)

Fig. 5 Fig. 6
Fig. 5 Photomicrographs of stained hepato-pancreas sections (H&E; scale bar 20μm).A Hepato-pancreas of the control group displaying normal histo-architectures of hepatic cells (arrow) and pancreatic acini (arrowhead).B Hepato-pancreas of the titanium dioxide nanogel (TDNG) group displays vacuolated hepatocytes (arrow) and preserved pancreatic acini (arrowhead).C The hepato-pancreas of the Pseudomonas putida group displays focal areas of degenerated and necrotic hepatocytes (arrow), a few apoptotic areas of pancreatic acini (arrowhead), and a congested portal vein (star).D Hepato-pancreas of the TDNG + P. putida group displays a dilated portal vein (star) with maintaining architectures of hepatocytes (arrow) and pancreatic acini (arrowhead)

Fig. 7 Fig. 8
Fig. 7 Photomicrographs of immunostained hepatopancreas sections (scale bar 20μm) for BCL-2 immunoreactivity.A & B Hepatopancreas of control and titanium dioxide nanogel (TDNG) groups displaying marked immune-staining level.C Hepatopancreas of Pseudomonas putida group displaying obvious reduction of immune-staining level.D Hepatopancreas of TDNG+P.putida group displaying mild to moderate immune-staining level

Fig. 9 Fig. 10
Fig. 9 Photomicrographs of immunostained hepatopancreas sections (scale bar 20μm) for caspase-3 immunoreactivity.A & B Hepatopancreas of control and titanium dioxide nanogel (TDNG) groups display undetectable immunostained cells.C Hepatopancreas of the Pseudomonas putida group displays diffuse cytoplasmic expressions in a wide number of hepatic cells.D Hepatopancreas of TDNG+P.putida group displays few positive immunostained cells

Table 1
The behavioral alterations of Nile tilapia experimentally infected with Pseudomonas putida and exposed to titanium dioxide nanogel (TDNG) (0.9 mg/L) for ten daysValues (means± SE) in the same row that do not share the same superscripts differ substantially (p < 0.05)